N-Glycan Profiles of Neuraminidase from Avian Influenza Viruses.

The cleavage of sialic acids by neuraminidase (NA) facilitates the spread of influenza A virus (IV) descendants. Understanding the enzymatic activity of NA aids research into the transmission of IVs. An effective method for purifying NA was developed using p-aminophenyloxamic acid-modified functionalized hydroxylated magnetic particles (AAMPs), and from 0.299 to 0.401 mg of NA from eight IV strains was isolated by 1 mg AAMP. A combination of lectin microarrays and MALDI-TOF/TOF-MS was employed to investigate the N-glycans of isolated NAs. We found that more than 20 N-glycans were identified, and 16 glycan peaks were identical in the strains derived from chicken embryo cultivation. Multi-antennae, bisected, or core-fucosylated N-glycans are common in all the NAs. The terminal residues of N-glycans are predominantly composed of galactose and N-acetylglucosamine residues. Meanwhile, sialic acid residue was uncommon in these N-glycans. Further computational docking analysis predicted the interaction mechanism between NA and p-aminophenyloxamic acid.

Alternations of N-glycans recognized by Phaseolus vulgaris leucoagglutinin in the saliva of patients with breast cancer.

Breast cancer is the most frequently diagnosed cancer in most countries. Early diagnosis of breast disease is necessary for its prognosis and treatment. Altered protein glycosylation has been shown to be expressed in precursor lesions of breast cancer, making them powerful early diagnostic biomarkers. The present study validated alterations of the N-glycan profiles of their salivary glycoproteins isolated by the Phaseolus vulgaris leucoagglutinin (PHA-E+L)-magnetic particle conjugates from 141 female subjects (66 healthy volunteers (HV), and 75 patients with breast disease including breast benign cyst (BB) or breast cancer in stage I/II (BC-I/II)) were analyzed and annotated by MALDI-TOF/TOF-MS. The results showed that there were 11, 20, 16, and 17 N-glycans recognized by PHA-E+L identified and annotated from the pooled salivary samples of HV, BB, BC-I, and BC-II, respectively. There were 3 N-glycans peaks (m/z 2459.8799, 2507.9139, and 2954.0547), 2 N-glycans peaks (m/z 1957.7265 and 2794.0427), and 2 N-glycans peaks (m/z 1866.6608 and 2240.8056) recognized by PHA-E+L that existed only in BB, BC-I, and BC-II, respectively. The present study compared the alternations of N-glycans from the salivary proteins isolated by PHA-E+L-magnetic particle conjugates among HV, BB, BC-I, and BC-II, which could provide information on N-glycans during the development of breast cancer in saliva to promote the study of its biomarkers.

Purification of sialoglycoproteins from bovine milk using serotonin-functionalized magnetic particles and their application against influenza A virus.

Sialylation is involved in receptor-ligand interactions, communication between cells, and host-pathogen interactions and it is involved in the ability of glycoproteins of bovine milk to inhibit the influenza A virus (IAV). The present paper describes a simple and efficient method to isolate sialoglycoproteins from bovine milk using serotonin-magnetic particle conjugates. Then, the isolated glycoproteins were analysed by lectin blotting and LC-MS/MS. The N-glycans on isolated glycoproteins were characterized by MALDI-TOF/TOF-MS. The role of the isolated sialoglycoproteins against IAV was validated in vitro. As a result, there were 91 proteins and 17 sialylated N-glycans to be identified. The isolated proteins have ability to inhibit attachment of IAV mimics to MDCK cells. However, the role of inhibition was abolished when the sialic acid moieties were destroyed. This method could provide useful information for the large-scale production of sialoglycoproteins from bovine milk against IAV.

Remarkable Homeostasis of Protein Sialylation in Skeletal Muscles of Hibernating Daurian Ground Squirrels (Spermophilus dauricus).

As the most common post-translational protein modification, glycosylation is intimately linked to muscle atrophy. This study aimed to investigate the performance of protein glycosylation in the soleus muscle (SOL) in Daurian ground squirrels (Spermophilus dauricus) and to determine the potential role of protein glycosylation in the mechanism underlying disuse muscle atrophy prevention. The results showed that (1) seven glycan structures comprising sialic acid α2-3 galactose (SAα2-3Gal) were altered during hibernation; (2) alterations in the SAα2-3Gal structure during hibernation were based on changes in the expression levels of beta-galactoside alpha-2 and 3-sialyltransferases; and (3) α2-3-linked sialylated modifications of heat shock cognate 70 and pyruvate kinase and expression of 14-3-3 epsilon protein were oscillatorily changed during hibernation. Our findings indicate that the skeletal muscles of hibernating Daurian ground squirrels maintain protein sialylation homeostasis by restoring sialylation modification during periodic interbout arousal, which might protect the skeletal muscles against disuse atrophy.

Abnormal Galactosylated-Glycans recognized by Bandeiraea Simplicifolia Lectin I in saliva of patients with breast Cancer.

Currently, the definitive diagnosis in breast cancer requires biopsy and histopathology, such the most effective markers are tissue-based. However, the advantages of saliva in collection and storage make it possible for assessing human pathology and contributing to the development of cancer-related biomarkers for clinical application. The present study validated alteration of salivary protein glycopatterns recognized by Bandeiraea simplicifolia lectin I (BS-I) in the saliva of patients with breast diseases using saliva microarrays, and the N/O-glycan profiles of their salivary glycoproteins isolated by the BS-I-magnetic particle conjugates from 259 female subjects (66 healthy volunteers (HV), 65 benign breast cyst or tumor patients (BB), 66 patients with breast cancer in stage I (BC-I) and 62 patients with breast cancer in stage II (BC-II)) were analyzed by MALDI-TOF/TOF-MS. The results showed that the expression level of galactosylated glycans recognized by BS-I was significantly increased in patients with breast cancer compared with HV (p < 0.05). Totally, there were 11/10, 10/19, 7/24 and 7/9 galactosylated N-/O-linked glycans were identified and annotated from the pooled salivary samples of HV, BB, BC-I and BC-II, respectively. One galactosylated N-glycan peak (m/z 2773.977), and 4 galactosylated O-glycan peaks (m/z 868.295, 882.243, 884.270 and 1030.348) were found only in BC-I. These findings could provide pivotal information on galactosylated N/O-linked glycans related to breast cancer, and promote the study of biomarkers for early-stage breast cancer based on precise alterations of galactosylated N/O-glycans in saliva.

Alterations in serum protein glycopatterns related to small cell lung cancer, adenocarcinoma and squamous carcinoma of the lung.

Background: The main reason why lung cancer has maintained a high rate of morbidity and mortality is that its early diagnosis is difficult. No current lung cancer screening is recommended by any major medical organization due to the lack of sensitive and specific screening technologies. Thus, this study aimed to systematically investigate the correlation between the alterations in serum glycosylation and three main types of lung cancers (SCLC, ADC and SqCC). Materials and methods: We investigated the protein glycopatterns in sera from 333 subjects (65 healthy volunteers, 38 benign lung disease patients, 49 small cell lung cancer patients, and 181 NSCLC patients) using a lectin microarray. A serum microarray was produced to evaluate and verify the terminal carbohydrate moieties of the glycoproteins in individual serum samples from 30 cases simultaneously. Results: There were 16 lectins (e.g., RCA120, BS-I, and UEA-I), 24 lectins (e.g., HHL, PTL-I, and MAL-II), and 18 lectins (e.g., GSL-I, LEL, and ACA) that exhibited significant differences in serum protein glycopatterns in the patients with SCLC, ADC and SqCC compared with the controls (HV and BPD). There were 6 lectins (e.g., EEL, NPA, and LEL) that exhibited significantly increased NFIs in ADC and SqCC compared with SCLC. Also, there were 5 lectins (e.g., Jacalin, BS-I, and UEA-I) that exhibited significantly decreased NFIs in ADC compared with SCLC and SqCC. Conclusions: This study can facilitate the discovery of potential biomarkers for the differential diagnosis of lung cancer based on the precise alteration in serum protein glycopatterns.

Analysis of Glycosphingolipid Glycans by Lectin Microarrays.

Glycosphingolipids (GSLs) are ubiquitous glycoconjugates of cell membranes. Identification of unknown GSL-glycan structures is still a major challenge. To address this challenge, we developed a novel strategy for analysis of GSL-glycans from cultured cells based on a lectin microarray that can directly detect and reveal glycopatterns of GSL extracts without the need for glycan release. There were six steps to perform the analysis of GSL-glycans: (i) extraction of GSLs from cell pellets, (ii) quantification of GSL-glycans using orcinol-sulfuric acid reaction, (iii) preparation of lyso-GSLs by using sphingolipid ceramide N-deacylase, (iv) fluorescence labeling of lyso-GSLs, (v) detection by a lectin microarray, (vi) data acquisition and analysis. Simultaneously, a supplementary verification analysis for GSL-glycans was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Optimized experimental conditions, which consisted of the blocking buffer, incubation buffer, and appropriate GSL concentration, were investigated by analyzing the glycopatterns of a standard ganglioside (GM1a) via lectin microarray. The analysis of GSL-glycans from human hepatocarcinoma cell lines (MHCC97L, MHCC97H, and HCCLM3) showed that there were 27 lectins (e.g., WFA, MAL-II, and LTL) to give significantly different signals compared with a normal human liver cell line (HL-7702), indicating up- and/or down-regulations of corresponding glycopatterns such as α1-2 fucosylation and α2-3 sialylation, and changes of certain glycostructures such as Galß1-3GalNAcß1-4(NeuAcα2-3)Galß1-4Glc:Cer and GalNAcα1-3(Fucα1-2)Galß1-3GlcNAcß1-3Galß1-4Glc:Cer. The lectin microarray analysis of lyso-GSLs labeled by fluorescence has proven to be credible, which can provide the glycopatterns and detailed linkage information on GSL-glycans.

Intact Glycopeptide Analysis of Influenza A/H1N1/09 Neuraminidase Revealing the Effects of Host and Glycosite Location on Site-Specific Glycan Structures.

Influenza H1N1 virus has posed a serious threat to human health. The glycosylation of neuraminidase (NA) could affect the infectivity and virulence of the influenza virus, but detailed site-specific glycosylation information of NA is still missing. In this study, intact glycopeptide analysis is performed on an influenza NA (A/H1N1/California/2009) that is expressed in human 293T and insect Hi-5 cells. The data indicate that three of four potential N-linked glycosylation sites are glycosylated, including one partial glycosylation site from both cell lines. The NA expressed in human cells has more complex glycans than that of insect cells, suggesting the importance of selecting an appropriate expression system for the production of functional glycoproteins. Different types of glycans are identified from different glycosites of NA expressed in human cells, which implies the site-dependence of glycosylation on NA. This study provides valuable information for the research of influenza virus as well as the functions of viral protein glycosylation.

Increased expression of core-fucosylated glycans in human lung squamous cell carcinoma.

Lung cancer is the most frequent cancer and the leading cause of cancer around the world. As one of the major types of lung cancer, lung squamous cell carcinoma (LUSC) is closely associated with smoking and shows poor sensitivity to therapy and prognosis. Although alteration of glycopatterns are reliable indicators of cancer, little is known about the alterations of protein glycosylation related to LUSC. In this study, we compared the differential expression levels of glycopatterns in seven pairs of LUSC tissues and normal pericarcinomatous tissues (PCTs) using lectin microarrays. Fluorescence-based lectin histochemistry and lectin blotting were utilized to validate and assess the expression and distribution of certain glycans in LUSC tissues and PCTs. And we further analyzed their total N-linked glycans using MALDI-TOF/TOF-MS to provide more information about the aberrant glycopatterns. The results showed that the expression level of the core fucosylation recognized by Pisum sativum agglutinin (PSA) and Lens culinaris agglutinin (LCA) was significantly increased in LUSC tissues compared with PCTs. There were 10 and 15 fucosylated N-linked glycans that were detected in PCTs and LUSC tissues respectively, 10 fucosylated N-glycans were common, while five fucosylated N-glycans were unique to LUSC tissues. And the abundance of the fucosylated N-glycans was increased from 40.9% (PCTs) to 48.3% (LUSC). These finding is helpful to elucidate the molecular mechanisms underlying the lung diseases and develop new treatment strategies.

The Function of Fucosylation in Progression of Lung Cancer.

Lung cancer is a disease that influences human health and has become a leading cause of cancer mortality worldwide. However, it is frequently diagnosed at the advanced stage. It is necessary by means of biology to identify specific lung tumor biomarkers with high sensitivity. Glycosylation is one of the most important post-translational modifications and is related to many different diseases. It is involved in numerous essential biological processes, such as cell proliferation, differentiation, migration, cell-cell integrity and recognition, and immune modulation. However, little was known about deregulation of glycosylation in lung cancer and contribution to tumor-microenvironment interactions. Among the numerous glycosylations, fucosylation is the most common modification of glycoproteins and glycosylated oligosaccharides. Increased levels of fucosylation have been detected in various pathological conditions, as well as in lung cancer. In this article, we reviewed the role of fucosylation in lung cancer. We highlighted some of the fucosylation alterations currently being pursued in sera or tissues of lung cancer patients. Moreover, we elaborated on the regulation mechanism of fucosylation in proliferative invasion and metastasis of lung tumor cells. In summary, alterations in fucosylation provide potential biomarkers and therapeutic targets in lung cancer.

Genetic variation and co-evolutionary relationship of RNA polymerase complex segments in influenza A viruses.

The RNA polymerase complex (RNApc) in influenza A viruses (IVs) is composed of the PB2, PB1 and PA subunits, which are encoded by the three longest genome segments (Seg1-3) and are responsible for the replication of vRNAs and transcription of viral mRNAs. However, the co-evolutionary relationships of the three segments from the known 126 subtypes IVs are unclear. In this study, we performed a detailed analysis based on a total number of 121,191 nucleotide sequences. Three segment sequences were aligned before the repeated, incomplete and mixed sequences were removed for homologous and phylogenetic analyses. Subsequently, the estimated substitution rates and TMRCAs (Times for Most Recent Common Ancestor) were calculated by 175 representative IVs. Tracing the cladistic distribution of three segments from these IVs, co-evolutionary patterns and trajectories could be inferred. The further correlation analysis of six internal protein coding segments reflect the RNApc segments have the closer correlation than others during continuous reassortments. This global approach facilitates the establishment of a fast antiviral strategy and monitoring of viral variation.

N-glycan profiles in H9N2 avian influenza viruses from chicken eggs and human embryonic lung fibroblast cells.

N-glycosylation can affect the host specificity, virulence and infectivity of influenza A viruses (IAVs). In this study, the distribution and evolution of N-glycosylation sites in the hemagglutinin (HA) and neuraminidase (NA) of H9N2 virus were explored using phylogenetic analysis. Then, one strain of the H9N2 subtypes was proliferated in the embryonated chicken eggs (ECE) and human embryonic lung fibroblast cells (MRC-5) system. The proliferated viral N-glycan profiles were analyzed by a glycomic method that combined the lectin microarray and MALDI-TOF/TOF-MS. As a result, HA and NA of H9N2 viruses prossess six and five highly conserved N-glycosylation sites, respectively. Sixteen lectins (e.g., MAL-II, SNA and UEA-I) had increased expression levels of the glycan structures in the MRC-5 compared with the ECE system; however, 6 lectins (e.g., PHA-E, PSA and DSA) had contrasting results. Eleven glycans from the ECE system and 13 glycans from the MRC-5 system were identified. Our results showed that the Fucα-1,6GlcNAc(core fucose) structure was increased, and pentaantennary N-glycans were only observed in the ECE system. The SAα2-3/6Gal structures were highly expressed and Fucα1-2Galß1-4GlcNAc structures were only observed in the MRC-5 system. We conclude that the existing SAα2-3/6Gal sialoglycans make the offspring of the H9N2 virus prefer entially attach to each other, which decreases the virulence. Alterations in the glycosylation sites for the evolution and role of IAVs have been widely described; however, little is known about the exact glycan structures for the same influenza strain from different hosts. Our findings may provide a novel way for further discussing the molecular mechanism of the viral transmission and virulence associated with viral glycosylation in avian and human hosts as well as vital information for designing a vaccine against influenza and other human viruses.

Serum Glycopatterns as Novel Potential Biomarkers for Diagnosis of Acute-on-Chronic Hepatitis B Liver Failure.

Acute-on-chronic hepatitis B liver failure (ACHBLF) is an increasingly recognized distinct disease entity encompassing an acute deterioration of liver function in patients with cirrhosis, so little is known about the alterations of protein glycopatterns in serum with its development. We aimed to identify the alterations of serum glycopatterns in ACHBLF and probe the possibility of them as novel potential biomarkers for diagnosis of ACHBLF. As a result, there were 18 lectins (e.g., WFA, GSL-II, and PNA) to give significantly alterations of serum glycopatterns in ACHBLF compared with healthy controls (HC) (all p ≤ 0.0386). Meanwhile, among these lectins, there were 12 lectins (e.g., WFA, GAL-II, and EEL) also exhibited significantly alterations of serum glycopatterns in ACHBLF compared with HBV-infected chronic hepatitis (cHB) (all p ≤ 0.0252). The receiver-operating characteristic (ROC) curve analysis indicated there were 5 lectins (PHA-E + L, BS-I, ECA, ACA, and BPL) had the greatest discriminatory power for distinguishing ACHBLF and HC or cHB, respectively (all p ≤ 0.00136). We provided a new basic insight into serum glycopatterns in ACHBLF and investigated the correlation of alterations in serum glycopatterns as novel potential biomarkers for diagnosis of ACHBLF.

A pilot study of salivary N-glycome in HBV-induced chronic hepatitis, cirrhosis, and hepatocellular carcinoma.

Hepatitis B is a potentially life-threatening liver infection caused by the hepatitis B virus (HBV), which can lead to chronic liver disease and put people at high risk of death from cirrhosis of the liver and liver cancer. However, little is known about the correlation of salivary N-linked glycans related to HBV-infected liver diseases. Here we investigated N-linked glycome in saliva from 200 subjects (50 healthy volunteers (HV), 40 HBV-infected patients (HB), 50 cirrhosis patients (HC), and 60 hepatocellular carcinoma patients (HCC) using MALDI-TOF/TOF-MS. Representative MS spectra of N-glycans with signal-to-noise ratios >6 were annotated using the GlycoWorkbench program. A total of 40, 47, 29, and 33 N-glycan peaks were identified and annotated from HV, HB, HC, and HCC groups, respectively. There were 15 N-glycan peaks (e.g., m/z 1647.587, 1688.613 and 2101.755) were present in all groups. Three N-glycan peaks (m/z 2596.925, 2756.962, and 2921.031) were unique in HV group, 2 N-glycan peaks (m/z 1898.676 and 1971.692) were unique in HB group, 5 N-glycan peaks (m/z 1954.677, 2507.914, 2580.930, 2637.952, and 3092.120) were unique in HC group, and 3 N-glycan peaks (m/z 2240.830, 2507.914, and 3931.338) were unique in HCC group. The proportion of fucosylated N-glycans was apparently increased in the HCC group (84.8%) than in any other group (73.1% ± 0.01), however, the proportion of sialylated N-glycans was decreased in HCC group (12.1%) than in any other group (17.23% ± 0.003). Our data provide pivotal information to distinguish between HBV-associated hepatitis, cirrhosis and HCC, and facilitate the discovery of biomarkers for HCC during its early stages based on precise alterations of N-linked glycans in saliva.

Alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV.

The incidence of hepatocellular carcinoma (HCC) is closely correlated with hepatitis B virus (HBV)-induced liver cirrhosis. Structural changes in the glycans of serum and tissue proteins are reliable indicators of liver damage. However, little is known about the alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV infection. This study compared the differential expression of liver glycopatterns in 7 sets of normal pericarcinomatous tissues (PCTs), cirrhotic, and tumor tissues from patients with liver cirrhosis and HCC induced by HBV using lectin microarrays. Fluorescence-based lectin histochemistry and lectin blotting were further utilized to validate and assess the expression and distribution of certain glycans in 9 sets of corresponding liver tissue sections. Eight lectins (e.g., Jacalin and AAL) revealed significant difference in cirrhotic tissues versus PCTs. Eleven lectins (e.g., EEL and SJA) showed significant alteration during cirrhotic and tumor progression. The expression of Galα1-3(Fucα1-2)Gal (EEL) and fucosyltransferase 1 was mainly increasing in the cytoplasm of hepatocytes during PCTs-cirrhotic-tumor tissues progression, while the expression of T antigen (ACA and PNA) was decreased sharply in cytoplasm of tumor hepatocytes. Understanding the precision alteration of liver glycopatterns related to the development of hepatitis, cirrhosis, and tumor induced by HBV infection may help elucidate the molecular mechanisms underlying the progression of chronic liver diseases and develop new antineoplastic therapeutic strategies.

Differentially expressed glycosylated patterns of α-1-antitrypsin as serum biomarkers for the diagnosis of lung cancer.

Lung cancer is the most common malignancy worldwide. Thus, there is a critical need for diagnostic biomarkers with adequate sensitivity and specificity for lung cancer detection. Glycans in glycoproteins are significantly altered in cancer, and may serve as a tool for identifying potential diagnostic biomarkers. Recent studies have reported changes in α-1-antitrypsin (A1AT) glycosylation in lung cancer serum, tissue and cell lines. In this study, a lectin microarray was used to detect glycosylation changes in serum A1AT from patients with lung adenocarcinoma (ADC), squamous cell lung cancer, small-cell lung cancer (SCLC) and benign pulmonary diseases. Differentially expressed glycosylated patterns of A1AT were identified by lectin arrays and were confirmed by lectin-based enzyme-linked immunosorbent assay (ELISA). We found that galactosylated A1AT could distinguish non-small-cell lung cancer (NSCLC) from benign pulmonary diseases (AUC = 0.834); fucosylated A1AT showed exceptional capability in distinguishing ADC from benign diseases (AUC = 0.919) or other lung cancer subtypes (AUC = 0.844), and A1AT containing poly-LacNAc could detect SCLC from benign diseases (AUC = 0.905) or NSCLC (AUC = 0.707). The present study indicates that glycosylated patterns of A1AT may serve as potential biomarkers for detection of lung cancer. Further studies in larger sample sizes are necessary to validate the clinical utility of these markers.

Profiling of concanavalin A-binding glycoproteins in human hepatic stellate cells activated with transforming growth factor-ß1.

Glycoproteins play important roles in maintaining normal cell functions depending on their glycosylations. Our previous study indicated that the abundance of glycoproteins recognized by concanavalin A (ConA) was increased in human hepatic stellate cells (HSCs) following activation by transforming growth factor-ß1 (TGF-ß1); however, little is known about the ConA-binding glycoproteins (CBGs) of HSCs. In this study, we employed a targeted glycoproteomics approach using lectin-magnetic particle conjugate-based liquid chromatography-tandem mass spectrometry to compare CBG profiles between LX-2 HSCs with and without activation by TGF-ß1, with the aim of discovering novel CBGs and determining their possible roles in activated HSCs. A total of 54 and 77 proteins were identified in the quiescent and activated LX-2 cells, respectively. Of the proteins identified, 14.3% were glycoproteins and 73.3% were novel potential glycoproteins. Molecules involved in protein processing in the endoplasmic reticulum (e.g., calreticulin) and calcium signaling (e.g., 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase ß-2 [PLCB2]) were specifically identified in activated LX-2 cells. Additionally, PLCB2 expression was upregulated in the cytoplasm of the activated LX-2 cells, as well as in the hepatocytes and sinusoidal cells of liver cirrhosis tissues. In conclusion, the results of this study may aid future investigations to find new molecular mechanisms involved in HSC activation and antifibrotic therapeutic targets.

Selective isolation and analysis of glycoprotein fractions and their glycomes from hepatocellular carcinoma sera.

As one of the most important post-translational modifications, the discovery, isolation, and identification of glycoproteins are becoming increasingly important. In this study, a Con A-magnetic particle conjugate-based method was utilized to selectively isolate the glycoproteins and their glycomes from the healthy donor and hepatocellular carcinoma (HCC) case sera. The isolated glycoproteins and their N-linked glycans were identified by LC-ESI-MS/MS and MALDI-TOF/TOF-MS, respectively. A total of 93 glycoproteins from the healthy donors and 85 glycoproteins from the HCC cases were identified. There were 34 different glycoproteins shown between the healthy donors (21/34) and the HCC cases (13/34). Twenty-eight glycans from the healthy donors and 30 glycans from the HCC cases were detected and there were 22 different glycans shown between the healthy donors (10/22) and HCC cases (12/22). Among these glycoproteins, 50 were known to be N-linked glycoproteins and three novel glycopeptides from two predicted potential glycoproteins were discovered. Moreover, lectin blotting, Western blotting and lectin/glyco-antibody microarrays were applied to definitely elucidate the change of selective protein expressions and their glycosylation levels, the results indicated that the differences of the identified glycoproteins between the healthy donors and HCC cases were caused by the change of both protein expression and their glycosylation levels.

Isolation and identification of native membrane glycoproteins from living cell by concanavalin A-magnetic particle conjugates.

The discovery, isolation, and subsequent identification of cell membrane glycoproteins involved in the structure and function of the cell surface are becoming more and more important. Here, concanavalin A-magnetic particle conjugates were employed to isolate the special membrane glycoproteins from living HepG-2 cells. The isolated glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry as well as annotated. A total of 37 membrane glycoproteins were identified, and 25 of them were ascertained to locate in the extracellular region.